Oral candidias is a biofilm based fungal infection that can have severe implications particularly in the growing population of immunocompromised patients. Despite their clincal importance, very little research has been done on fungal biofillms including candida. Few if any studies involving in situ analysis of C. albincans biofilms have been published. One ex situ study has shown that Candida biofilms, like those of bacteria, are resistant to antimicrobial agents. Although a variety of mechanisms have been proposed to explain the recalcitrance of bacterial biofilms to antibiotics, the mechanisms by which Candida biofilms resist the action of antifungal agents are unknown. The goal of this proposed research is to delineate the relationship between the structure of C. albicans biofilms and their resistance to antimicrobial agents. In this study a combination of in situ methods will be used to quantitatively investigate the structure of C. albicans biofilms. These methods will include Attenuated Total Reflectance Infrared Spectroscopy (ATR/FTIR), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), electrochemical microsensors, fluorescent microscopy and scanning laser confocal microscopy. The influence of a variety of environmental factors including fluid shear, growth media, serum and saliva proteins and substratum chemistry on the biofilm structure will be investigated. These factors will then be used to manipulate the biofilm structure in order to determine the influence of structural features on the antifungal resistance of the biofilms. The efficacy of three antimicrobial agents, hydrogen peroxide, chlorhexidine gluconate, and fluconazole, against both biofilms and planktonic C. albicans will be measured. In addition, penetration of these agents into the biofilm will be measured with a combination of ATR/FTIR, ToF-SIMS imaging of cryosections, and microelectrodes. Multivariate statistical modeling will be used to identify relationships between the biofilm structure and antimicrobial resistance. Mechanistic differential equation based models will be used to identify possible causal relationships between biofilm structure and antifungal efficacy.